The Src family kinase (SFK) is a subfamily of non-receptor tyrosine kinases. SFK members, Src and especially Fyn, are expressed in the striatum. These SFK members are involved in the regulation of neuronal and synaptic activities and are linked to the pathogenesis of a variety of neuropsychiatric and neurodegenerative disorders. Given the fact that muscarinic acetylcholine (mACh) receptors are highly expressed in striatal neurons and are critical for the regulation of striatal function, we investigated the role of mACh receptors in the regulation of SFKs in the adult rat striatum in vivo. We found that pharmacological blockade of mACh receptors by systemic administration of the mACh antagonist scopolamine induced a marked increase in phosphorylation of SFKs in the striatum of male and female rats. This scopolamine-induced increase in SFK phosphorylation occurred in the two subdivisions of the striatum (caudate putamen and nucleus accumbens) and was time-dependent and reversible. Another mACh antagonist atropine was also effective in stimulating SFK phosphorylation. Coadministration of subthreshold doses of scopolamine and a dopamine D1 receptor agonist SKF81297 enhanced striatal SFK phosphorylation. Between Fyn and Src proteins immunoprecipitated from striatal tissue, scopolamine selectively increased phosphorylation of Fyn. The increase in Fyn phosphorylation was accompanied by an increase in Fyn kinase activity in response to scopolamine. These results reveal a significant role of mACh receptors in the regulation of SFKs (mainly Fyn) in striatal neurons. Under normal conditions, endogenous mACh receptors appear to exert an inhibitory effect on Fyn activity.